In the previous program project grant we examined the functions of the B7-CD28/CTLA4 co-stimulatory pathway in patients with MS. In this renewal, we will build on our studies to investigate: 1) Are there defects of immune regulation in the clonal expansion and cytokine secretion of autoreactive T cells as a consequence of B7 engagement of CTLA-4? We hypothesize that ICOS stimulation influences the generation of regulatory T cells in autoimmune disease and can lead to down-regulation of an autoimmune response via production of inhibitory cytokines, particularly IL-10. We will examine (2) expression of ICOS & ICOS counter-receptor on T cells and APCs isolated from blood and CNS tissue of MS patients and control. 93) We will compare the functional consequences of ICOS and CD28-mediated co-stimulatory signals on the proliferative and cytokine responses of naive and activated T cells at the whole population and single cell level. Cytokine expression will be measured by intracytoplasmic staining and gene chip technology Single cell cloning of ICOS positive and negative T cells will examine the functional role of T cells expressing ICOS. We will determine the effects of ICOS crosslinking on naive vs. Th1 or Th2 polarized T cells to determine whether ICOS signaling can de-differentiate a Th1 cytokine response; and (4) we will determine whether peripheral blood T cells from patients with MS or diabetes have defects in the ICOS pathway leading to dysregulation of the immune response. Using blockade with ICOS-Ig or stimulation with antigen with transfectants expressing MHC and ICOS- counter-receptor, we will examine both autoantigen specific T cells and the invariant Valpha24JalphaQ T cells which are defective in Th2 cytokine secretion in patients with autoimmune disease. Together, these studies will provide fundamental information regarding the role of ICOS and B7/CD28/CTLA-4 in regulating T cell activation and differentiation in MS and IDDM patients and lead to new approaches for the treatment of autoimmune disease.